Additive systems for use in protein PEGylation

ABSTRACT

The present disclosure provides an additive system for use in protein PEGylation. The additive system includes p-aminobenzoic hydrazide used either alone or in combination with aromatic amines, such as 3,5-diaminobenzoic acid, or with ammonium salts such as ammonium chloride or ammonium acetate. The disclosed additive combination provides several benefits including increased reaction rates, higher yields and reduction in the aminoxy-PEG equivalents required to complete the conjugation reaction. Typical reactions can be run by combining the additive or additive system with a solution of a protein and aminoxy-PEG reagent. The solution is adjusted to pH 4 and held at 20-25° C. without stirring until completion, typically within 24 hours.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent Ser. No.15/776,923 filed May 17, 2018, now allowed, which is a 35 U.S.C. § 371National Stage patent application of International ApplicationPCT/US2016/063313, filed Nov. 22, 2016, which claims priority to U.S.Provisional Application Ser. No. 62/258,644, filed Nov. 23, 2015; theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to an improved additive system for use inprotein PEGylation reaction. In particular, the disclosure identifies anadditive for the conjugation reaction between proteins containing thep-acetylphenylalanine residue and an aminoxy-PEG compound.

BACKGROUND

The PEGylation of proteins is a conjugation process that involves theattachment of a polyethylene glycol derivative to a therapeutic proteinto improve its stability and pharmacokinetics by reducing clearancerates and providing a steric shield from proteolytic enzymes and immunesystem recognition (Roberts, M. J. et al., Adv. Drug Delivery Rev,54:459 (2002)). In general, the PEGylation technologies can beclassified into two types, namely random and site-specific conjugations.Random PEGylations arbitrarily link the PEGylating reagent to reactiveamino acids such as lysine or cysteine to afford a mixture of PEGylatedproducts. In contrast, site-specific conjugations exploit theunambiguous reactivity of a native functionality (e.g., the N- orC-terminal groups) or an unnatural amino acid (e.g.,p-acetylphenylalanine—pAcF) to control the location and number of PEGresidues attached to the protein. Site specific conjugation reactioninvolving ketoxime formation between a PEGylating reagent and a pAcFresidue is incorporated in the substrate protein via expansion of thegenetic code (Liu, C. C. et al., Annu. Rev. Biochem., 79:413 (2010);Tian, F. et al., “Accelerants for the modification of non-natural aminoacids and non-natural amino acid polypeptides”, U.S. Pat. No. 7,468,458(Dec. 23, 2008)). Despite their and demonstrated utility, conjugationsbased on the formation of ketoximes suffer from slow rates andincomplete conversions (Crisalli, P. et al., J Org. Chem., 78:1184(2013)). Attempts to improve ketoxime formation include the use ofexcess PEGylating reagent, high temperatures, or high concentrations oftoxic catalysts. These solutions, however, introduce additional steps toeliminate the excess PEGylating reagent or toxic catalyst from theproduct and often compromise the stability of the protein. Additionally,the old methods do not use additives, use a denaturant (urea), and/oruse acetylhydrazide (AcNHNH2) as the additive. AcNHNH2 and relatedstructures have been defined in PCT Publication No. WO 2007/056448.

What is now needed in the art are new methods to upgrade the yield andrates for the PEGylation of proteins (Relaxin and FGF21) containing apAcF residue by examining the mechanistic principles that effectstalling and by identifying new additives that accelerate the reactionand promote high conversions at low PEG: protein molar ratios. Themethods should be economical, promote higher conversions withconsiderably lower amounts of PEGylating reagent, promote fasterreactions that circumvent the need for high reaction temperatures andelimination of genotoxic material.

SUMMARY

In a first embodiment, the present disclosure provides an improvedadditive system for protein PEGylation reaction, said system comprisingp-aminobenzoic hydrazide alone or in combination with aromatic amines orammonium salts.

In another embodiment, the present disclosure provides a process forobtaining PEGylated protein, said process comprising steps of:identifying a protein, PEG reagent and an additive system; andsolubilizing the protein followed by combining with PEG reagent inpresence of the additive system to obtain PEGylated protein with highyield.

In a further embodiment, the present disclosure provides apharmaceutical composition comprising a PEGylated protein obtained bythe process as mentioned in the above embodiment for use in therapy fora subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: General mechanism for the formation of ketoximes.

FIG. 2: Chromatogram for the PEGylation of Relaxin (4 mg/mL in water)with 20 kDa PEG-OA reagent.

FIG. 3: Time course for the decomposition of 20 kDa PEG-OA in water whenexposed to a continuous stream of air.

FIG. 4: Study of the stability of acetyl hydrazide using in situ IR and¹H NMR spectra.

FIG. 5: Model reaction for the screening of additives. Reactionconditions: 1 (3.6 mmol) and 2 (3.6 mmol) in 1.0 mL acetate buffer (20mM, pH 4.0) at room temperature (23° C.).

FIG. 6: Relative rates (k_(rel)) observed for different additives.Acetyl hydrazide (k_(rel)≈2) is shown in a box.

FIG. 7: Time course for the reaction of dipeptide 1 withO-benzylhydroxylamine (2) in the presence of (a) 1 equiv pyrazoleamine(red); (b) 1 equiv MCH (blue); (c) 1 equiv pyrazoleamine and 1 equiv MCH(green). The reaction profile obtained in the absence of additives isshown in grey.

FIG. 8: Aromatic region of the ¹H NMR spectra of samples containingdipeptide 1 (a) (blue) with (b) 1 equiv MCH (green); (c) 1 equiv MCH,and 1 equiv pyrazoleamine (grey); (d) 1 equiv pyrazoleamine (red).Synergistic effect between MCH and pyrazoleamine additives yieldsmixtures with higher concentration of active intermediates relative tosamples containing only one additive.

FIG. 9: Left: PEGylation of the dipeptide 1 with 30 equiv PABH and 1.2equiv 20 kDa PEG-OA; the hydrazone intermediate is tracked as the greenpoints. Right: PEGylation reaction in which 1.2 equiv PEGylating reagentwere added after equilibrating the dipeptide with 30 equiv PABHovernight.

FIG. 10: Left: Plot of hydrazone concentration versus equivalents ofPABH for the PEGylation of dipeptide 1; the blue points indicate thereaction mixture in which pyrazoleamine was omitted. Center: Effect ofdifferent combinations of PABH and pyrazoleamine on reaction rates.Right: Final concentration of dipeptide 1 and its hydrazone derivativein reaction mixtures containing PABH and pyrazoleamine.

FIG. 11: Plot of remaining dipeptide 1 at the end of the reaction,versus total equivalents additive and pyrazoleamine: PABH ratio.

FIG. 12: Left: Time course for the reaction of Relaxin with 20 kDaPEG-OA (1.5 equiv) in the presence of (a) 30 equiv acetyl hydrazide(blue); (b) 30 equiv MCH (red); the reaction profile obtained in theabsence of additives is shown in grey color. Center: Time course for thereaction of Relaxin with PEG-OA (1.5 equiv) in the presence of (a) 30equiv pyrazoleamine (blue); (b) 30 equiv MCH (red); (c) 10 equiv MCH(purple); (d) 30 equiv pyrazoleamine and 10 equiv MCH (green); (e) 30equiv MCH and 30 equiv pyrazoleamine. Right: Time course for thereaction of Relaxin with PEG-OA (1.5 equiv) in the presence of 30 equivpyrazoleamine and 10 equiv MCH at (a) 40° C.; (b) 10° C.; (c) 25° C.

FIG. 13: Left: Time course for the reaction of Relaxin with 20 kDaPEG-OA (1.2 equiv) in the presence of (a) 30 equiv MPCH and 30 equivpyrazoleamine (green); (b) 30 equiv PH and 30 equiv pyrazoleamine(blue); and (c) 30 equiv MCH and 30 equiv pyrazoleamine in urea 6M(red). Right: Time course for the reaction of Relaxin with PEG-OA (1.2equiv) in the presence of (a) 30 equiv PH and 60 equiv pyrazoleamine(blue); (b) 30 equiv acetyl hydrazide and 60 equiv pyrazoleamine(green); and (c) 30 equiv PABH and 60 equiv pyrazoleamine (red).

FIG. 14: HRMS analysis of a PEGylation of Relaxin accelerated by MCH atits endpoint. The oxime peak overlaps with the residual Relaxin. The0.04 min delay for the Relaxin peak is due to the reaction matrix effecton chromatographic behavior rather than a late-eluting impurity.

FIG. 15: Left: Time course for the reaction of Relaxin with 20 kDaPEG-OA (1.2 equiv) in the presence of urea 6M with (a) 30 equiv MCH and30 equiv pyrazoleamine (red); (b) 30 equiv MCH, 30 equiv pyrazoleamine,and 30 equiv NH₂OH (blue). Right: Time course for the decay of Relaxinwith PEG-OA (1.2 equiv) in the presence of urea 6M with 30 equiv MCH and30 equiv pyrazoleamine (blue); the oxime peak (red) grows during thereaction, suggesting parallel formation of hydroxylamine from PEG-OAdecomposition.

FIG. 16: Left: Time course for the reaction of Relaxin with 20 kDaPEG-OA (1.2 equiv) in the presence of 30 equiv PABH and (a) 60 equivethylenediamine (grey); (b) 60 equiv 3,5-diaminobenzoic acid (green);(c) 60 equiv m-phenylenediamine (blue); and 60 equiv pyrazoleamine(red). Right: Time course for the reaction of Relaxin with PEGOA (1.2equiv) in the presence of (a) 30 equiv PABH and 60 equiv3,5-diaminobenzoic acid (green); and (b) 60 equiv PABH and 120 equivNH₄Cl (blue).

FIG. 17: Scheme for the preliminary PEGylation screening.

FIG. 18: Scheme for the PEGylation of Relaxin and FGF21 with differentamines.

FIG. 19: PEGylation results for o- and m-phenylenediamine illustratingthe curvature of the plot of total equiv versus amine:PABH ratio versusfinal FGF21 concentration. Left: o-phenylenediamine. Right:m-phenylenediamine.

FIG. 20: Plots of total additive equivalents versus amine:PABH ratio andconversion for PEGylations using PABH and 3,5-diaminobenzoic acid. Left:FGF21 with 30 kDa PEG-OA. Right: Relaxin with 20 kDa PEG-OA.

FIG. 21: Left: Time course for the reaction of Relaxin with 20 kDaPEG-OA (1.2 equiv) in the presence of 120 equiv salt and 60 equiv PABHcatalyst using (a) urea 6 M (green); (b) NH₄Cl (grey); and (c)(NH₄)₂SO₄.

FIG. 22: Time course for the reaction of Relaxin with 20 kDa PEG-OA (1.2equiv) in the absence of additives (blue) and the presence of: (a) 120equiv NH₄Cl (red), (b) 60 equiv acetyl hydrazide (grey), and (c) 60equiv acetyl hydrazide and 120 equiv NH₄Cl (green).

FIG. 23: Left: UV spectra of Relaxin before and after the addition of120 equiv NH₄Cl (blue and red, respectively). Right: Amino acid sequenceof Relaxin highlighting the aromatic resides in red.

FIG. 24: Left: IR spectra of Relaxin before and after the addition of120 equiv NH₄Cl (blue and purple, respectively). Right: Inset of the IRspectrum after addition of 120 equiv NH₄Cl with tentative assignmentsfor the structural changes.

FIG. 25: ¹⁵N NMR HSQC spectra of Relaxin before and after the additionof 120 equiv NH₄Cl (red and blue, respectively).

FIG. 26: Near UV CD spectra of Relaxin before (blue) and after theaddition of 120 equiv NH₄Cl (blue and green, respectively).

FIG. 27: Structures of potential impurities present in commercial PABH.

DETAILED DESCRIPTION

Unless otherwise specifically set forth elsewhere in the application,the following terms may be used herein, and shall have the followingmeanings.

Abbreviations

PEG: polyethylene glycol

PEG-OA: polyethylene glycol-oxyamine

mPEG: methoxy polyethylene glycol

MCH: morpholine 4-carbohydrazide

MPCH: 4-methylpiperazine-1-carbohydrazide

PH: Pivalic hydrazide

PABH: p-amino benzoic hydrazide

PMBH: p-methoxy benzoic hydrazide

It must be noted that as used herein, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art. Unless otherwise stated, all ranges describedherein are inclusive of the specific endpoints. The following terms areprovided below.

About: The term “about” is used herein to mean approximately, roughly,around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 5 percent up or down(higher or lower).

Additive system: The term “additive system” is used herein to mean“catalyst compound”, either alone or in combination. For, e.g.,p-aminobenzoic hydrazide alone or in combination with aromatic aminesnamely 3,5-diaminobenzoic acid, 0-phenylenediamine,1-pyridin-2-yl-ethylamine, 2-(dimethylamino)ethylhydrazine,m-phenylenediamine and 2-picolylamine or ammonium salts namely ammoniumacetate and ammonium chloride. Preferable catalyst compounds includesp-aminobenzoic hydrazide with 3,5-diaminobenzoic acid or p-aminobenzoichydrazide with ammonium chloride. Comprising: The term “comprising”means “including”, e.g., a composition “comprising” X may consistexclusively of X or may include something additional, e.g., X+Y.

PEG: The term “PEG” when used in the context of this disclosure refersto polyethylene glycol or derivatized polyethylene glycol.

PEGylation or pegylation process: The term “PEGylation” or “pegylationprocess” refers to the process of attachment of polyethylene glycol(PEG) polymer chains to another molecule, in the context of the presentdisclosure, to proteins containing p-acetylphenylalanine (pAcF) residueincluding, but not limited to, Relaxin and FGF21.

Conjugation: The term “conjugation” as used herein refers to aconjugation reaction between proteins containing p-acetylphenylalanineresidue and an aminoxy-PEG compound.

It generally involves the activation of PEG and coupling of theactivated PEG-intermediates directly to target proteins/peptides or to alinker, which is subsequently activated and coupled to targetproteins/peptides (see Abuchowski, A. et al., J. Biol. Chem., 252:3571(1977) and J. Biol. Chem., 252:3582 (1977), Zalipsky et al. inPoly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications, Chapters 21 and 22, Harris, J. M., ed., Plenum Press, NY(1992)). It is noted that a polypeptide containing a PEG molecule isalso known as a conjugated or PEGylated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated orfree.

PEG reagent or PEGylating reagent: Reagents that help in PEGylationreaction.

It will be understood that any given exemplary embodiment can becombined with one or more additional exemplary embodiments.

In a first aspect, the present disclosure provides an improved additivesystem for protein PEGylation reaction, said system comprisingp-aminobenzoic hydrazide alone or in combination with aromatic amines orammonium salts.

In a first embodiment of the first aspect, the aromatic amine isselected from a group consisting of 3,5-diaminobenzoic acid,O-phenylenediamine, 1-pyridin-2-yl-ethylamine, 2-(dimethylamino)ethylhydrazine, m-phenylenediamine and 2-picolylamine.

In a second embodiment of the first aspect, the ammonium salt isselected from a group consisting of ammonium acetate and ammoniumchloride.

In a third embodiment of the first aspect, the preferred systemcombination includes p-aminobenzoic hydrazide with 3,5-diaminobenzoicacid or p-Aminobenzoic hydrazide with ammonium chloride.

In a fourth embodiment of the first aspect, the reaction is aconjugation reaction between proteins containing thep-acetylphenylalanine reside and an aminoxy-PEG compound.

In a fifth embodiment of the first aspect, the additive system augmentsthe conjugation reaction rates, provides high yield of the conjugatedproduct and facilitates reduction in the aminoxy-PEG equivalentsrequired to complete the conjugation reaction.

In a second aspect, the present disclosure provides a process forobtaining PEGylated protein, said process comprising steps of:identifying a protein, PEG reagent and an additive system; andsolubilizing the protein followed by combining with PEG reagent inpresence of the additive system to obtain PEGylated protein with highyield.

In a first embodiment of the second aspect, the protein is Relaxin orFGF21 containing a pAcF residue.

In a second embodiment of the second aspect, the solubilized proteinsolution combined with PEG reagent is maintained at a pH of about 4.

In a third embodiment of the second aspect, the reaction mixture is heldat a temperature ranging from about 20° C. to about 25° C.

In a fourth embodiment of the second aspect, the additive systemincludes p-aminobenzoic hydrazide alone or in combination withcombination with aromatic amines, such as 3,5-diaminobenzoic acid orammonium salts such as ammonium acetate or ammonium chloride.

In a fifth embodiment of the second aspect, the additive combination ofhigh quality p-aminobenzoic hydrazide with ammonium chloride ispreferred for use at large scale production of PEGylated proteins.

In a sixth embodiment of the second aspect, the PEG reagents areselected from a group comprising PEG-OA and other PEG derivatives withaminoxy group.

In a third aspect, the present disclosure provides a pharmaceuticalcomposition comprising a PEGylated protein obtained by the process ofrecited in the second aspect and its embodiments for use in therapy fora subject in need thereof.

EXAMPLES

The present disclosure will now be described in connection with certainembodiments which are not intended to limit its scope. On the contrary,the present disclosure covers all alternatives, modifications, andequivalents as can be included within the scope of the claims. Thus, thefollowing Examples, which include specific embodiments, will illustrateone practice of the present disclosure, it being understood that theExamples are for the purposes of illustration of certain embodiments andare presented to provide what is believed to be the most useful andreadily understood description of its procedures and conceptual aspects.

The general mechanism of the reaction between a carbonyl group and ahydroxylamine derivative is well-understood for small molecule reactants(Jencks, W. P., Prog. Phys. Org. Chem., 2:63 (1964), and referencescited therein). The process is acid-catalyzed and, overall, entails adehydration preceded by a multistep equilibrium. While ketimines areformed at slower rates than aldimines due to allylic 1,3-strain, forsimple alkyloxyamines the ketone-ketimine equilibrium is largely shiftedtowards dehydration (FIG. 1).

Example 1 Study to Investigate the Decomposition of the PEGylatingReagent as a Potential Cause for Reaction Stalling

One possible cause for stalling of the PEGylation could result fromdecomposition of the PEGylating reagent. As the PEG reagent is not UVactive it requires a different detection method to monitor its fateduring the reaction. Evaporative light scattering detection involvespassing the HPLC mobile phase through a nebulizer to remove the solvent.Any solid particles that form diffract light from a laser beam in thedetector, resulting in a signal. This method allows detection of anycompound that forms a solid that can diffract light. As the PEGylatingreagents are high molecular weight solids, they are excellent candidatesfor HPLC analysis using ELS detection. This is evident in FIG. 2. The UVtrace is shown in green, the ELS trace is shown in black. The topchromatogram (green) is the UV trace at 210 nm, the black chromatogramis the ELS trace of the same mixture obtained in series with the UVdetector. Clearly the bottom black trace provides more information,particularly with the later eluting PEG-based compounds.

The latest eluting peak is not UV active and was not present at thestart of the reaction. This suggested competitive decomposition of thePEGylating reagent. Indeed, this compound formed when the solution ofPEGylating reagent was exposed to air (FIG. 3). The conversion of the 20kDa PEG-OA reagent was >97% at concentrations≈4 mg/mL and resulted inbyproducts that were not reactive in the PEGylation.

As this decomposition is the result of reaction of PEGylating reagentwith dissolved gas in the solvent, there should be an inversecorrelation between decomposition rates and concentrations. Thishypothesis matches the observation that samples of 20 kDa PEG-OA at morereaction relevant concentrations of 30-40 mg/mL were quite stable (<5%decomposition). While there may be some impact to analytical work forthis project to ensure sample stability during analysis, the minimaldecomposition under more reaction relevant conditions lead to theconclusion that PEG decomposition by air is not a strong cause forobserved reaction stalling. The stability of the PEGylating reagent wasalso studied in the presence of additives employed to accelerate thePEGylation. These experiments were conducted at the expected reactionconcentrations, assuming the target PEG loading of 1.2 equiv relative toRelaxin. In all cases, the decomposition of the PEGylating reagent wasminimal.

Since PEGylation stalling occurs in the presence of a large excess ofacetyl hydrazide as an accelerating additive, the stability of thishydrazide under the reaction conditions was tested by in situ IR and ¹HNMR spectroscopies. The studies showed that acetyl hydrazide is stableand suggest that the excess required to promote the reaction is mostlikely related to the existence of equilibration and a modestacceleration relative to the uncatalyzed background process (FIG. 4).

Example 2 Studies on Screening Additives Using Dipeptide Model System(DMS)

Initial efforts to find additives that accelerated the reaction betweenthe pAcF ketone handle in Relaxin and an alkoxyamine involved thescreening of commercially available compounds that contained activatedX—NH₂ moieties capable of promoting the formation of their correspondingimino derivatives through dehydration. Minimum requirements for theselection of these additives were their sufficient stability andsolubility under the temperature, buffer and pH conditions used topromote the PEGylation of the actual protein in aqueous medium. Tosimplify analytical procedures and facilitate reaction monitoring byHPLC-UV and NMR spectroscopy, the condensation between dipeptideAla-pAcF (1) and O-phenylhydroxylamine (2, refer FIG. 5) was chosen as amodel transformation that could guide the additive choice. In asubsequent step, additives that improved the model reaction would betested in the PEGylation of Relaxin and FGF21. Provided in FIG. 5 is amodel reaction for the screening of additives. Reaction conditions: 1(3.6 mmol) and 2 (3.6 mmol) in 1.0 mL acetate buffer (20 mM, pH 4.0) atroom temperature (23° C.).

Reaction rates and percent conversion were evaluated for fifty additivesclassified in four general categories, namely: anilines, hydrazines,hydrazides and hydrazinecarboxamines depending on the nature of the Xsubstituent on the X—NH₂ moiety. Observed rates were normalized to therate measured in the absence of additives (k_(rel)=1). Acetyl hydrazide(k_(rel)≈2) was a reference additive to establish a baseline acceptableperformance; only those additives that afforded k_(rel)>2 and highconversions (>95%) would be considered for subsequent application andoptimization in the PEGylation of Relaxin and FGF21. A summary of theresults is shown in FIG. 6. The reactions were carried out in HPLC vialsat room temperature without stirring to mimic protein PEGylationconditions, and aliquots were periodically drawn by the HPLC autosampler to avoid further sample manipulation.

Hydrazides (colored in blue) and hydrazinecarboxamides (green) providedthe best results. In general, anilines (red) afforded full dipeptideconversion but did not accelerate the reaction. Moreover, most anilinespromoted high levels of undesired epimerization. Hydrazines (yellow)formed large amounts of hydrazone under the reaction conditions and werediscarded for further study. Aromatic hydrazides and secondaryhydrazinecarboxamines yielded up to five-fold rate accelerations as wellas high conversions that stalled at approximately 95% of dipeptideconsumption. In particular, the screening led to the finding ofmorpholine-4-carbohydrazide (MCH, 4) and p-aminobenzoic hydrazide (PABH,7) as optimal reagents to promote the transformation. Based on theirperformance, solubility at pH 4, commercial availability and cost, PABHand MCH were further evaluated in the PEGylation of Relaxin and FGF21(vide infra).

With this information in hand, attempts to circumvent reaction stallingfocused on three aspects: (a) the addition of variable amounts ofadditive, (b) the effect of chaotropic agents, and (c) the combinationof additives. Initially, studies were performed with model dipeptide andacetyl hydrazide to ascertain intrinsic trends in reactivity. Monitoringreaction rates at varying amounts of acetyl hydrazide revealedsaturation kinetics in additive, indicating that the rate enhancementreaches a threshold value at high concentrations of reagent (Scheme 1).

Moreover, growing concentrations of acetyl hydrazide promoted higherstalling levels in agreement with a general mechanism involving amultistep sequence of reversible imine transfer reactions. In thepresence of an accelerating additive, such mechanism pointed towards thepossibility of achieving better conversions by shifting the apparentequilibrium towards dehydration. Empirical attempts to modify thisequilibrium through changes in the reaction medium were unsuccessful.For example, the addition of 6M urea or NH₄Cl did not accelerate thereaction nor affected the original conversion levels. The screening ofcertain anilines resulted in complete conversion without accelerationand envisioned that the combination of these anilines with anaccelerating additive could help extend substrate conversion. Indeed,the use of a mixture of acetyl hydrazide and pyrazoleamine 9 led tofaster reaction profiles and nearly complete conversions (FIG. 6). ¹HNMR analysis of mixtures containing equimolar amounts of dipeptide,pyrazoleamine, MCH additive, or a mixture of MCH and pyrazoleaminerevealed a synergistic growth of the imine and hydrazone intermediatesfor the samples containing the mixture and suggested that the positiveeffect of combining an accelerating additive and an aniline correlateswith a shift of the equilibrium en route to the reaction intermediates(FIG. 8).

Analogous ¹H NMR spectroscopic studies using PABH, an additive thatcontains both the hydrazide and aniline moieties, show the prevalentformation of a single hydrazone intermediate in agreement with DFTcalculations. Under neutral conditions, computations at theB3LYP/6-31G(d) level of theory favor the formation of the hydrazonebetween pAcF and PABH rather than its isostructural imine by ≈3 kcal/mol(Scheme 2).

Protonation of the amino acid —NH₂ (pKa≈9), supports hydrazone formationby 9 kcal/mol. Double protonation (pKa: aniline≈2.5, hydrazide <2)favors imine formation by ≈1.0 kcal/mol. Although the reaction is notrun at such low pH values, DFT calculations indicate that thedehydration is highly sensitive to pH variations and H-bonding effects.

The hydrazone intermediate could be monitored during the PEGylation ofthe dipeptide 1 with PABH (FIG. 9). Control experiments thatequilibrated mixtures of dipeptide 1 with PABH overnight in the absenceof PEGylating reagent showed the formation of the hydrazone as well asits rapid consumption upon addition of 20 kDa PEG-OA to give the desiredproduct. Further examination of hydrazone formation in the presence ofamine additives (e.g., pyrazoleamine, FIG. 10) suggested that the extentof hydrazone formation is linked to the pyrazoleamine: PABH ratio usedin the reaction. Interestingly, the extent of hydrazone formation doesnot correlate with reaction rates or conversions in a simple manner. Inthe model dipeptide system, a pyrazoleamine: PABH ratio of 1:1 wasoptimal for formation of the hydrazone. However, in the case of reactionrates, optimum conditions corresponded with a 1:2 ratio amine:hydrazide.The need for higher amounts of hydrazide relative to amine is supportedby the preliminary screening in which hydrazides were observed toprovide much higher reaction rates than the amine additives. On theother hand, a 3:1 ratio of amine:hydrazide was optimal for dipeptideconversion consistent with (a) the observation of significant amounts ofhydrazone intermediate remaining at the end of the reaction at lowamine:hydrazide ratio and (b) the reversal of the reaction detected uponaddition of “kicker charges” of the hydrazide additive. In theexperiments presented above, the total equivalents are the sum of thePBAH and pyrazoleamine. The effect of changes in the total equivalentsof additive was addressed via a series of experiments run varying boththe amine:hydrazide ratio as well as the total equivalents of combinedadditives. The results are summarized in FIG. 11. The curvature in thefigure suggests that the cooperative effect between the amine andhydrazide is complex and optimization of the reaction conditions mayrequire consideration of not only the total equivalents of theadditives, but the ratio between the two.

Example 3 Additive Accelerated PEGylations of Proteins—Relaxin and FGF21

Application of the lessons learned in the model system to the PEGylationof Relaxin and FGF21 aimed to decrease the number of equivalents of 20kDa PEG-OA to a maximum of 1.2 equiv as well as shortening reactiontimes at room temperature without compromising reaction yields orquality of the product. Towards this end, tested the additivesidentified in the model reaction with Relaxin under reaction conditionsinitially developed from cursory investigations. PEGylation of Relaxin(21 mg/mL in 20 mM AcONa at pH 4.0) with 1.5 equiv PEG-OA afforded goodconversions (≈90%) after 24 h at room temperature in the presence of 30equiv acetyl hydrazide or MCH. In the absence of catalyst, underidentical conditions, the reaction gave significantly lower conversions(≈75%). Consistent with the model system studies, the PEGylation wasaccelerated by the additives, and the reaction with MCH was two-foldfaster than the reaction with acetyl hydrazide. Moreover, theequilibration proposed in FIG. 6 found support in the followingobservations: (a) catalyzed reactions stalled at comparable conversions,(b) once stalled, addition of 30 equiv extra additive decreasedconversion levels, and (c) once stalled, addition of 0.5 equiv PEG-OAtook the reaction to higher conversion (Z 95%). Trends noticed foranilines in the model reaction translated to the PEGylation of Relaxin:the addition of 30 equiv pyrazoleamine transformed most of the Relaxinstarting material without acceleration and, combining MCH withpyrazoleamine yielded 95% conversion in only 8 h. Efforts to optimizethe reaction conditions exposed a complex interplay between thevariables and encouraged the application of DoE studies to gain deeperinsight (Example 4). For example, higher or lower amounts of MCH mixedwith 30 equiv pyrazoleamine did not improve conversion, and the use ofhigher temperatures did not promote faster reactions (FIG. 12).Decreasing the charge of PEG-OA to 1.2 equiv caused stalling at ≈85-90%conversion under the conditions optimized for 1.5 equiv in the presenceof the additives MCH, MPCH (5) or PH (6). However, in contrast with itsnegligible effect in the model system, the use of MCH in urea 6Madvanced reaction completion up to ≈95% (FIG. 12).

Advancement of the PEGylation to completion, however, uncovered theformation of an impurity with the same retention time as the Relaxinstarting material but unreactive in the presence of PEGylating reagent.HRMS studies indicated that the impurity corresponded to the oxime atthe pAcF N-terminal residue and spiking NH₂OH confirmed that theimpurity was unproductive towards PEGylation (FIG. 13). Two hypotheseswere postulated to explain the source of NH₂OH, namely: (a) its presenceas an input impurity in the PEGylating reagent, and (b) its formationduring the course of the PEGylation reaction. Careful analysis of thePEG-OA demonstrated that NH₂OH levels in the starting materials were<0.05 ppm and disproved the first hypothesis. In agreement withdegradation of the PEGylating reagent during the reaction, monitoringthe formation of the oxime impurity showed its unambiguous growththroughout the PEGylation (FIG. 14). A systematic HRMS study ofadditives 4-9 indicated that MCH and MPCH promoted decomposition of thePEG-OA, whereas acetyl hydrazide, PH, PABH, PMBH, and pyrazoleamine didnot. Consequently, in the optimization of PEGylations mediated by PABHsince PABH affords conversions comparable to those of MCH (FIG. 6) andits cost is much lower than the latter (1 U$/g vs 60 U$/g). Optimizedconditions involved the use of 30 equiv PABH and 60 equiv pyrazoleamine.These results would be subsequently confirmed by DoE studies.

A revision of the effect of anilines upon reaction conversion observedduring the screening in the model system showed that, in addition topyrazoleamine, three amines were able to advance completion levels.These were m-phenylenediamine, ethylenediamine, and 3,5-diaminobenzoicacid. Using 1.2 equiv PEG-OA and 30 equiv PABH, the addition of 60 equivamine afforded conversions ≈95% (FIG. 19). Lower additivestoichiometries resulted in lower conversions at short times (≈90%) butslowly achieved higher conversions at 24 h.

Example 4 DoE Studies to Evaluate the Interactions Between DifferentAdditive Combinations

The interplay between amine and hydrazide additives was further exploredfor the PEGylation of Relaxin and FGF21 using a DOE approach that tookinto account four variables, namely (a) the identity of the hydrazide,(b) the identity of the amine, (c) total equivalents of additives, and(d) amine:hydrazide ratio of the additives used. The studies wereperformed with Relaxin and FGF21 to examine the effect of theamine:hydrazide molar ratio and PEG-OA loadings on rates andconversions, and analyze a variety of amines previously identified inthe model system (FIG. 6). The results of these experiments pointed to acomplex interplay between the amine:hydrazide ratio and indicated thatreaction optimization requires careful consideration of the type ofamine as well as the substrate protein.

In the first round of screening (Table 1), we utilized PABH, acetylhydrazide, and pivalic hydrazide in combination with pyrazoleamine forthe PEGylation of both Relaxin and FGF21 (FIG. 17). PEG-OA equivalentsand urea concentration were also considered. Most of the additivesystems in this screen gave good performance. In agreement with previousstudies (FIGS. 7 and 12), the 2:1 amine:hydrazide system was among thebest for promoting high conversion. Although pivalic hydrazide was anexcellent hydrazide additive that could promote high conversion and fastreaction rates, new impurities were observed in the reaction mixturesthat were not present when PABH was used.

TABLE 1 Results from Preliminary Screening of the PEGylation of RelaxinPyrazoleamine Hydrazide Conversion (equiv) Hydrazide (equiv) (%) 60Pivalic hydrazide 30 98 60 PABH 30 96  0 Pivalic hydrazide 60 96 30Acetyl hydrazide 10 96

This screen was repeated using FGF21 with similar results (Table 2).PABH proved to be the standout additive to accelerate the reaction and,as one of the goals of this initiative was to develop a generalPEGylation method that can be applied to abroad range of proteinsystems, PABH was selected for further study. Additional support for theuse of PABH as the hydrazide component was the fact that it is negativein AMES testing unlike acetyl hydrazide, which is a known potent mutagen(Bhide, S. V. et al., Cancer Lett., 23:235 (1984)).

TABLE 2 Results from the Preliminary Screening of the PEGylation ofFGF21 Pyrazoleamine Hydrazide Conversion (equiv) Hydrazide (equiv) (%) 0PABH 30 91 60 PABH 60 89 30 PABH 30 88 30 Pivalic hydrazide 60 83 10Acetyl hydrazide 60 82 60 Pivalic hydrazide 10 80 0 Acetyl hydrazide 1077 10 Pivalic hydrazide 10 73 0 Acetyl hydrazide 30 69

Having chosen PABH as the hydrazide, it was screened in combination witha broader scope of amines, using both Relaxin and FGF21 proteins (Scheme3). For these experiments the following considerations were made: (a)amines previously tested in the model system (FIG. 5) would be screened,(b) total additive equivalents (sum of hydrazide and amine) would rangefrom 20 to 120, and (c) reaction times would be limited to 24 h.

As the first DOE screening was conducted using PABH and pyrazoleamine,it was of primary interest to determine which of the amines (or class ofamines) was optimum for use in additive systems with PABH. Experimentalresults from the screening with Relaxin and FGF21 are summarized below(Tables 3 and 4, respectively).

TABLE 3 Data from Relaxin DoE Round 2 Con- Amine:PABH Total Time versionAmine Additive Ratio Equiv (h) (%) o-Phenylenediamine 0.6 20 22.7 96o-Phenylenediamine 1.7 50 21.0 96 o-Phenylenediamine 0.3 85 18.5 94o-Phenylenediamine 0.6 120 18.4 94 1-Pyridin-2-yl- 0.3 20 22.2 94ethylamine 1-Pyridin-2-yl- 3.0 20 23.7 93 ethylamine 1-Pyridin-2-yl- 0.3120 22.6 91 ethylamine 1-Pyridin-2-yl- 3.0 120 23.5 88 ethylamine2-(Dimethyl- 0.3 20 23.1 94 amino)ethylhydrazine 2-(Dimethyl- 3.0 2023.1 88 amino)ethylhydrazine 2-(Dimethyl- 1.0 85 21.4 84amino)ethylhydrazine 2-(Dimethyl- 3.0 120 19.7 55 amino)ethylhydrazine3,5-Diaminobenzoic acid 3.0 20 18.8 94 3,5-Diaminobenzoic acid 1.0 5020.1 95 3,5-Diaminobenzoic acid 3.0 120 21.8 94 3,5-Diaminobenzoic acid0.3 120 23.5 94 m-Phenylenediamine 3.0 20 19.3 94 m-Phenylenediamine 0.685 18.9 94 m-Phenylenediamine 3.0 120 23.1 96 m-Phenylenediamine 0.3 12019.7 93 p-Phenylenediamine 1.0 20 19.3 96 p-Phenylenediamine 3.0 50 18.996 p-Phenylenediamine 1.6 120 20.6 94

TABLE 4 Data from FGF21 Round 2 Con- Amine:PABH Total Time version AmineAdditive Ratio Equiv (h) (%) o-Phenylenediamine 0.64 120 23.5 60o-Phenylenediamine 3.00 85 23.7 70 o-Phenylenediamine 0.33 85 24.3 57o-Phenylenediamine 1.74 50 20.9 67 o-Phenylenediamine 0.64 20 20.5 64m-Phenylenediamine 3.00 20 24.4 48 m-Phenylenediamine 0.33 120 24.9 55m-Phenylenediamine 0.33 20 24.1 70 m-Phenylenediamine 0.64 85 24.7 37m-Phenylenediamine 3.00 120 20.9 75 3,5-Diamino- 3.00 20 23.9 55 benzoicacid 3,5-Diamino- 0.33 120 23.2 58 benzoic acid 3,5-Diamino- 0.33 2022.3 70 benzoic acid 3,5-Diamino- 1.00 50 20.1 76 benzoic acid3,5-Diamino- 3.00 120 19.7 77 benzoic acid Ethylenediamine 1.00 20 24.359 Ethylenediamine 3.00 50 24.0 55 Ethylenediamine 0.33 50 22.7 73Ethylenediamine 0.33 120 21.4 77 Ethylenediamine 1.57 120 20.5 522-Picolylamine 0.33 85 23.6 65 2-Picolylamine 3.00 85 23.1 742-Picolylamine 1.00 120 22.4 68 2-Picolylamine 1.57 20 24.5 582-Picolylamine 0.33 20 19.2 54 1-Pyridin-2-yl- 0.33 120 22.2 58ethylamine 1-Pyridin-2-yl- 3.00 120 23.1 42 ethylamine 1-Pyridin-2-yl-3.00 20 24.0 24 ethylamine 1-Pyridin-2-yl- 1.57 85 21.8 70 ethylamine1-Pyridin-2-yl- 0.64 50 23.2 68 ethylamine 1-Pyridin-2-yl- 0.33 20 20.153 ethylamine 2-(Dimethyl- 3.00 20 22.7 52 amino)ethylhydrazide2-(Dimethyl- 0.33 20 22.8 48 amino)ethylhydrazide 2-(Dimethyl- 0.33 12022.8 70 amino)ethylhydrazide 2-(Dimethyl- 3.00 120 19.7 20amino)ethylhydrazide 2-(Dimethyl- 1.00 85 19.3 49 amino)ethylhydrazide

The results for the PEGylation of Relaxin are consistent between thevarious amines, and conversions in general are reasonably high. On theother hand, for FGF21 3,5-diaminobenozic acid appears to be a goodpromoter of the PEGylation at both 1.0 and 3.0 amine:PABH ratios.Holding the total equivalents and ratio constant we get a better pictureof the impact the amine has on conversions. For Relaxin this data ispresented in Table 5.

TABLE 5 Effect of the Choice of Amine on Conversion for the PEGylationof Relaxin Con- Amine:PABH Total Time version Amine Additive Ratio Equiv(h) (%) 1-Pyridin-2-yl-ethylamine 0.33 120 28.1 91 m-Phenylenediamine0.33 120 25.2 94 1-Pyridin-2-yl-ethylamine 0.33 120 22.6 91p-Phenylenediamine 0.33 120 25.0 94 3,5-Diaminobenzoic acid 0.33 12023.5 93 2-(Dimethyl- 0.33 120 32.1 88 amino)ethylhydrazine

For Relaxin, the impact of the amine additive is apparent, but given thehigh overall conversions observed, this impact is again low. For FGF21,the impact is higher (Table 6).

TABLE 6 Effect of the Choice of Amine on Conversion for the PEGylationof FGF21 Amine:PABH Total Time Conversion Amine Additive Ratio Equiv (h)(%) m-Phenylenediamine 0.33 120 24.9 55 3,5-Diaminobenzoic acid 0.33 12023.2 58 Ethylenediamine 0.33 120 21.4 77 1-Pyridin-2-yl-ethylamine 0.33120 22.2 58 2-(Dimethylamino)ethyl- 0.33 120 22.8 70 hydrazide

From these data it is clear that conversion is highly dependent on theidentity of the amine used in combination with PABH. Realizing theseconditions do not necessarily reflect the optimum conditions for thePEGylation reaction, we sorted by conversion to arrive at the list ofthe top five performing additive combinations for FGF21 (Table 7).

TABLE 7 Top 5 Performing Additive Systems for the PEGylation of FGF21Total Amine:PABH Time Conversion Amine Additive Equiv Ratio (h) (%)m-Phenylenediamine 120 3.00 20.9 75 3,5-Diaminobenzoic acid 50 1.00 20.176 3,5-Diaminobenzoic acid 120 3.00 19.7 77 Ethylenediamine 120 0.3321.4 77 2-Picolylamine 85 3.00 23.1 74

Ethylenediamine and 3,5-diaminobenzoic acid were the two top performersfor the PEGylation of FGF21 under the conditions screened. As3,5-diaminobenzoic acid is a relatively inexpensive and readilyavailable crystalline solid, it was deemed a favorite additive for theamine:PABH combination. Further support will come from the detailedanalysis of total equivalents versus amine:PABH ratios. As observed inthe model system, the cooperative effect of the two additives is quitecomplex. In fact, the curvature observed in the model system was morepronounced for the PEGylation of Relaxin and FGF21. The data and trendsfor FGF21 are shown in FIG. 19. Anilines o- and m-phenylenediamine weregood additives when used in the PEGylation of FGF21. Interestingly, aplot of the final concentration of remaining protein versus the totalequivalents and amine:PABH ratio afforded completely different results.The only structural difference between these two additives is theorientation of the amino groups.

As the z-axis is the final concentration of FGF21 in the reactionmixture, the ideal conditions will have the lowest z-axis value in theplot. For a robust process that can allow for minor differences in thereagent charges, a flat plot or a plot with a valley or well is ideal.One additive that met this premise is 3,5-diaminobenzoic acid (FIG. 20).

When used in PEGylation of Relaxin, 3,5-diaminobenzoic acid alsoproduced high conversions in reasonable reaction times. The comparisonof FIG. 20 (left and right) demonstrates that increased conversions arelinked to the identity of the protein as well.

Example 5 Use of Simple Ammonium Salts

In view of the beneficial effect of amine additives at pH 4, we reckonedthat simple ammonium salts could also improve the PEGylation of Relaxin.Towards this end, a series of experiments with varying amounts of PABHand NH₄OAc or NH₄Cl were carried out that revealed the superiority ofthe combination of 60 equiv PABH and 120 equiv NH₄Cl to afford the bestconversion yet observed at 10 h (97%, Table 8). Interestingly,re-examination of the reaction of the model dipeptide in the presence ofNH₄Cl provided a profile that was identical to the profile observedwithout NH₄Cl, suggesting that the effect of NH₄Cl can be associated tochanges in the structure of Relaxin. Indeed, the addition of knownchaotropic reagents such as urea or (NH₄)₂SO₄ afforded comparableresults (FIG. 21). The positive effect of NH₄Cl seems general andreappears in the PEGylation of Relaxin without hydrazide additive or inthe presence of acetyl hydrazide (FIG. 22).

TABLE 8 Conversions for the Screening of Additive Amounts and SimpleAmmonium Salts Reaction Additive Salt Conversion (10 h) 1 30 equiv  60equiv 90% pNHPhHydrazide NH₄OAc 2 30 equiv 120 equiv 94% (×3)pNHPhHydrazide NH₄OAc 3 60 equiv  60 equiv 95% pNHPhHydrazide NH₄OAc 460 equiv 120 equiv 92% pNHPhHydrazide NH₄OAc 5 60 equiv 120 equiv 97%pNHPhHydrazide NH₄Cl 6 30 equiv  60 equiv 95% pNHPhHydrazide NH₄Cl

Spectroscopic studies seeking a better understanding of the origins ofthe NH₄Cl effect suggested that the salt modifies the conformation ofRelaxin in solution. While UV spectroscopic analysis could not detectvariations in the absorbance of the aromatic residues (260-290 nm) uponaddition of 120 equiv NH₄Cl (FIG. 23), IR spectroscopy indicated theexistence of structural modifications on the amide II band H-bonding(FIG. 24). Presumably, the failure of the UV analysis to detect changeson the aromatic residues can be traced to the distal location of aminoacids Tyr(Y), Phe(F), and Trp(W) in Relaxin, which largely exposes theresidues to solvent and lack any significant intramolecularinteractions.

Subtle modifications on the structure of Relaxin found further supporton ¹⁵N NMR HSQC spectroscopic studies consistent with protein foldinggoing to higher percentage of random coil upon addition of NH₄Cl (FIG.25) and near UV CD investigations that showed small differences in thefingerprint region of Relaxin's tertiary structure possibly due todimerization interface changes (FIG. 26).

Example 6 Typical Procedure and Results

Based on the studies reported in this memorandum, the combination ofPABH and NH₄Cl was recommended for use at larger scale. Results fromvarious protein systems are given in the following table.

TABLE 9 Protein/PEG-OA Conditions¹ Conversion (%) Relaxin/20 kDa PEG-OA30 equiv PABH, 120 equiv 94 NH₄Cl, 20° C. Relaxin/dPEG36-OA 30 equivPABH, 30° C. 90 FGF21 G1/30 kDa PEG-OA 30 equiv PABH, 120 equiv 88NH₄Cl, 20° C. FGF21 G2/30 kDa PEG-OA 30 equiv PABH, 120 equiv 98 NH₄Cl,20° C. ¹Conditions: 1.2 equiv PEG-OA reagent, 20° C. unless otherwiseindicated. Conversions were determined by HPLC assay after 18-24 hreaction time.

One consideration when using PABH in PEGylation reactions is the purityof the commercially available reagents is not equal across vendors. Useof 98% PABH on large scale resulted in an impurity peak co-eluting withthe desired product during the chromatography. Isolation of theseimpurities confirmed their structures to be the hydrazide and amideimpurities present in the PABH (FIG. 27).

Due to the high equivalents used in the reaction, even low levelimpurities in this reagent can have an impact. Thus, it is recommendedto pursue the highest quality PABH available.

Example 7 Typical Screening Procedure

The use of the additives is operationally simple. PEGylation of FGF21 G1with 30 kDa PEG-OA is given as a representative example.

TABLE 10 Reagents and their concentrations Items Amt (mg) Vol (mL) MWμmol FGF21 Solution 20.3 1.0 19585 1.04 PABH 4.7 151.17 31.2 NH₄Cl 6.753.49 125 30 kDa PEG-OA 39.0 31000 1.26Procedure

A solution of FGF21 (1.0 mL, 20.3 mg/mL, 1.04 μmol) in 20 mM NaOAc, 6Murea, with pH 4 was added to solid NH₄Cl (6.7 mg, 124.8 μmol) in a clean1.5 mL vial. The mixture was gently agitated until all the soliddissolved. In a separate vial, MPEG 30 kDa (39.0 mg, 1.26 μmol) and PABH(4.7 mg, 31.2 μmol) were combined. The protein solution from the firstvial was transferred to the second vial containing the PEGylatingreagent and PABH, and the mixture gently agitated until the solidsdissolved (ca 20 min). The pH was measured and the mixture adjusted topH 4 using 0.1M HCl if needed.

Typical reaction mixture are homogenous solutions, thus the reactionsolution was held at 20-25° C. without stirring. Reaction progress ismonitored by HPLC either using ELS or UV detection at 280 nm. Reactioncompletion is assessed by HPLC analysis versus an external standard.

Example 8 Comparison of Pegylation Conditions

The final goal of this initiative was to demonstrate the utility of thisnew PEGylation procedure versus the procedures in place at the time ofthe start of the project, as well as the “state of the art” conditionsin the literature. This is primarily to quantify the savings inexpensive PEGylating reagents available by use of the additivecombinations recommended. Table 11 includes the original conditions usedfor Relaxin and FGF21 G1 PEGylations as well as the PEGylations mediatedby the PABH additive.

TABLE 11 Comparison of PEGylation Conditions with Emphasis on PotentialCost Savings PEG T PABH NH₄Cl Conv Protein PEG (equiv) (° C.) (equiv)(equiv) (%) Notes Relaxin 20 kDa 1.5 50 N/A N/A 96 Original conditions.PEG-OA Relaxin 20 kDa 1.2 20 30 120 94 20% reduction in PEG-OA PEG withsimilar yield. FGF21 G1 30 kDa 2.5 20 N/A N/A 75 Original conditions.PEG-OA FGF21 G1 30 kDa 1.2 20 30 120 88 58% reduction in PEG-OA PEG plus13% increase in yield.

Potential reduction in cost for the Relaxin compound is purely relatedto the amount of the PEG 20 kDa reagent used, as the conversions andyields of both processes is comparable. However, for the PEGylation ofthe first generation FGF21 asset, the savings is quite dramatic. Morethan 50% reduction in the PEG loading combined with a 13% increase inreaction yield result in an overall 70% reduction in cost associatedwith its production. As the cost of PABH and NH₄Cl are both extremelylow relative to the PEGylating reagents, their use contributes minimallyto the overall cost of production. In the following table we compare thePABH conditions with those using acetyl hydrazide.

TABLE 12 Summary of Results of PEGylation of Various Protein Systems andComparison with Acetyl Hydrazide Protein Conditions¹ Conversion (%)Relaxin G1 ²No additives, 50° C. 97 ²No additives, 20° C. 75 ²30 equivAcNHNH₂, 90 no co-additive, 20° C. 30 equiv PABH, no co- 86 additive,20° C. 30 equiv PABH, 120 equiv 94 NH₄Cl, 20° C. Relaxin G2 Noadditives, 40° C. 70 No additives, 30° C. 70 30 equiv AcNHNH₂, no 80co-additive, 30° C. 30 equiv PABH, no co- 95 additive, 30° C. 30 equivPABH, NH₄Cl, 90 30° C. FGF21 G1 No additives, 20° C. 53 30 equivAcNHNH₂, 80 no co-additive, 20° C. 30 equiv AcNHNH₂, 82 120 equiv NH₄Cl30 equiv PABH, 120 88 equiv NH₄Cl, 20° C. FGF21 G2 30 equiv PABH, 120 98equiv NH₄Cl, 20° C. ¹All reactions were run using 1.2 equiv ofPEGylating reagent, unstirred at 20° C.. ²Reactions were run using 1.5equiv PEGylating reagent.

Some of the advantages of the additive system in the present disclosureare listed below:

-   1) Promotion of higher conversions with considerably lower amounts    of PEGylating reagent (1.2 equiv relative to 1.5-2.5 equiv), which    is extremely expensive. The below table provides a comparison that    highlights the improvements over the old method (standard    conditions).

Conditions Conversion Standard with with Molecule Conditions ConversionAdditives Additives Relaxin 1.5 equiv PEG 96% 1.2 equiv PEG 96% 50° C.,48 h 60 equiv p- NHBzNHNH₂ 120 equiv NH₄Cl 20° C., 10 h FGF21 2.5 equivPEG 75% 1.2 equiv PEG 82% Urea 6M 30 equivp- 20° C., 24 h NHBzNHNH₂ 120equiv NH₄Cl 20° C., 24 h FGF21- 1.6 equiv PEG 88% 1.2 equiv PEG 89% GEN230 equiv 60 equiv p- AcNHNH₂ NHBzNHNH₂ 20° C., 24 h 120 equiv NH₄Cl 20°C., 24 h

The combination of two additives greatly increases reaction rate andconversions, and allows for dramatic reduction of PEG loading versus theoriginal conditions.

-   2) Promotion of faster reactions that circumvent the need for high    reaction temperatures. Avoiding high temperatures diminishes    associated concerns about protein structural modification and    stability in the reaction.-   3) Substitution of acetylhydrazide (Ames positive) by PABH (Ames    negative in preliminary studies). Eliminates the use of a genotoxic    material and associated controls in the final product.

Pharmaceutical Compositions

The PEGylated proteins prepared in accordance with instant disclosuremay be further rendered suitable for injection by mixture or combinationwith an additional pharmaceutically acceptable carrier or vehicle bymethods known in the art. Among the pharmaceutically acceptable carriersfor formulating the products of the invention are saline, human serumalbum, human plasma proteins, etc. The invention also relates topharmaceutical compositions comprising a conjugate as described aboveand a pharmaceutically acceptable excipient and/or carrier. Suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like. The proteinconjugates prepared in accordance with instant disclosure may beformulated in pharmaceutical compositions suitable for injection with apharmaceutically acceptable carrier or vehicle by methods known in theart. See, e.g., WO 97/09996, WO 97/40850, WO 98/58660, and WO 99/07401(each of which is hereby incorporated by reference in its entirety).

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing disclosure and that it can be embodiedin other specific forms without departing from the essential attributesthereof. It is therefore desired that the instant disclosure beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing disclosure, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

We claim:
 1. A process for obtaining PEGylated FGF21 protein, theprocess comprising a step of reacting FGF21 protein with a PEG reagentin presence of an additive system to obtain PEGylated FGF21 protein,wherein the additive system includes p-aminobenzoic hydrazide andwherein the PEG reagent is selected from a group consisting ofPEG-oxyamine and other PEG derivatives with an aminoxy group.
 2. Theprocess of claim 1, wherein the FGF21 protein carries ap-acetylphenylalanine residue.
 3. The process of claim 2, wherein thePEG reagent has the structure of formula (I)


4. The process of claim 3, wherein the PEGylated FGF21 protein has thestructure of formula (II)


5. The process of claim 4, wherein the FGF21 protein is reacted with thePEG reagent in the presence of the additive system at a pH of about 4.6. The process of claim 4, wherein the FGF21 protein is reacted with thePEG reagent in the presence of the additive system at a temperatureranging from about 20° C. to about 25° C.
 7. The process of claim 2,wherein the FGF21 protein is reacted with the PEG reagent in thepresence of the additive system at a pH of about
 4. 8. The process ofclaim 2, wherein the FGF21 protein is reacted with the PEG reagent inthe presence of the additive system at a temperature ranging from about20° C. to about 25° C.
 9. The process of claim 1, wherein the additivesystem includes p-aminobenzoic hydrazide in combination with an aromaticamine, such as 3,5-diaminobenzoic acid, or in combination with anammonium salt, such as ammonium acetate or ammonium chloride.
 10. Theprocess of claim 4, wherein the FGF21 protein is solubilized prior toreacting the FGF21 protein with the PEG reagent in the presence of theadditive system.
 11. The process of claim 2, wherein the FGF21 proteinis solubilized prior to reacting the FGF21 protein with the PEG reagentin the presence of the additive system.
 12. The process of claim 10,wherein the FGF21 protein is solubilized in urea prior to reacting theFGF21 protein with the PEG reagent in the presence of the additivesystem.
 13. The process of claim 11, wherein the FGF21 protein issolubilized in urea prior to reacting the FGF21 protein with the PEGreagent in the presence of the additive system.